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ABSTRACT: Retinoic acid (RA) induced differentiation of SH-SY5Y cells increases the expression of mu opioid receptors (HMOR) and inhibitory G proteins, as well as the efficacy of opioids to inhibit forskolin-induced adenylyl cyclase activity. We examined the time course of the effects of all-trans retinoic acid (RA) on HMOR and c-fos mRNA levels as determined by solution hybridization (using HMOR and rat c-fos riboprobes) in RNA extracts from SH-SY5Y cells. Electrophoretic Mobility Shift Assay (EMSA) and Western blot analysis were used to assess the changes AP-1 DNA binding and the presence of fos-related proteins in nuclear extracts from untreated, vehicle (ethanol) or RA-treated SH-SY5Y cells. Exposure to RA for 0.5 h had no effect on HMOR while after 6-18 h of exposure HMOR in mRNA levels were decreased by 50% and then after 168 h of RA exposure, HMOR mRNA levels were doubled. In contrast, c-fos mRNA levels were unchanged at 0.5 h, but increased by 50% after 18 and 168 h of RA exposure. RA increased AP-1 binding after 18 and 168 h and a pan fos-FRA antibody produced a supershift. Western analysis indicates that RA activates a 45-kDa protein corresponding to the size of the fos B protein. These results identify two signal transduction targets that are regulated by RA during differentiation.
Molecular Brain Research 03/2002; 99(1):34-9. · 2.00 Impact Factor
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ABSTRACT: Currently, 1.8 million Americans use cocaine, 30% of whom are females. Sex differences in the pattern of cocaine abuse may reside in neuroendocrinological modulations that affect the use of and/or dependence on cocaine. This review discusses sex differences in cocaine-induced behavioral and molecular alterations in the central nervous system, with emphasis on the role of endocrine responses in the neuronal modulations of this drug. Mechanisms and data supporting the role of the hypothalamic-gonadal axis in the modulation of cocaine-induced behavioral and molecular alterations are also provided.
Annals of the New York Academy of Sciences 05/2001; 937(1):140 - 171. · 3.15 Impact Factor
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ABSTRACT: We studied the effects of 10 μg of 17-β-estradiol-3-benzoate treatment on preproenkephalin (PPE) mRNA expression in female ovariectomized (OVX) Swiss Webster mice after 0, 1, 6, 12, 24, or 48 h, using the in situ hybridization technique. The VMH showed a 1.6- and 3.3-fold increase in PPE mRNA levels after 24 and 48 h of estrogen treatment (respectively) when compared to OVX females. No differences at 1, 6 or 12 h of estrogen treatment groups were observed compared to control groups. PPE mRNA levels were also increased at 24 and 48 h after estrogen treatment in the posterior medial nucleus of the amygdala (MeAmyg) by 3.3- and 2.5-fold, respectively, and in the arcuate nucleus (ARC) by 2- and 1.9-fold, respectively. No effects of estrogen were observed on PPE mRNA levels in the caudate-putamen (CPu) or the posterior lateral cortical nucleus of the amygdala (plCoAmyg). Furthermore, basal levels of PPE mRNA expression in the VMH and MeAmyg of female mice were lower than those observed in rats, although levels in the CPu, plCoAmyg, and ARC were similar between females of the two species. In conclusion, we have found two differences between the species. First, Swiss mice demonstrated a slower time course of estrogen induction of PPE mRNA in the VMH, ARC, and MeAmyg compared to female rats. Second, there are differences in basal levels of PPE in the MeAmyg and VMH.
Journal of Chemical Neuroanatomy 12/1996; · 2.43 Impact Factor
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ABSTRACT: Phosphodiester antisense oligodeoxynucleotides (ODNs) directed against various domains of the cloned mouse δ receptor DOR-1 reduce δ-opioid receptor binding in vivo and in vitro. The present study examines the stability of an antisense ODN (275 nM) directed against the δ-opioid receptor and its effect on DOR-1 mRNA in cultured neuroblastoma cells and in vivo. When added to NG108-15 cells, much of the antisense ODN is degraded. However, >1% is intact, associated with cells, and stable for at least 72 h. Northern blot analysis demonstrates that treatment of NG108-15 cells with the antisense ODN reduces the levels of a species of DOR-1 mRNA by ∼25%. Similarly, intrathecal administration of the antisense ODN results in the accumulation of intact ODN within the spinal cord, which is stable for at least 72 h, although the levels of accumulation in vivo are lower than in vitro after either 4 or 72 h. Antisense ODN treatment lowers DOR-1 mRNA levels by ∼25%. The loss of mRNA both in vivo and in vitro corresponds quite well to the decreases in receptor binding previously observed by our laboratory and is consistent with reduction of δ-opioid receptor protein in vitro as determined by western blot with a monoclonal antibody selective for the δ-opioid receptor. In conclusion, these studies indicate that a small, but significant, proportion of ODN is taken up by cells and remains intact for up to 72 h. This appears to be sufficient to down-regulate mRNA levels of δ-opioid receptors and their expression.
Journal of Neurochemistry 10/1995; 65(5):1981 - 1987. · 4.06 Impact Factor
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Marina Brodsky, Kathryn Elliott, Alexandra Hynansky, Shirzad Jenab, Charles E. Inturrisi
Neuroreport 01/1995; 6(5):725-729. · 1.66 Impact Factor
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ABSTRACT: Previous studies have suggested that opioids play a role in the regulation of reproductive behaviors in the female rat. The present study examined whether estrogen treatment alters μ-opioid receptor mRNA levels in different areas of the forebrain of ovariectomized (OVX) female rats using the in situ hybridization technique. We observed an increase in μ-opioid receptor mRNA levels in the ventromedial nucleus of the hypothalamus (VMH) and arcuate nucleus (ARN) after 48 h of 10 μg of 17-β-estradiol-3-benzoate treatment when compared to OVX females. No effects of estrogen were observed on μ-opioid receptor mRNA levels in the posterior medial nucleus of the amygdala (MeAmyg), hippocampus, caudate-putamen (CPu) or the medial habenula. Our result suggests that the estrogenic regulation of μ-opioid receptor in the CNS may in part be mediated by de novo synthesis and/or stability of the μ-opioid receptor message.
Molecular Brain Research.
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ABSTRACT: The effects of chronic treatment with naltrexone, an opioid receptor antagonist, on δ1- and δ2-opioid receptor agonist-induced antinociception and ligand binding were investigated in mice. Antinociception by intracerebroventricular (i.c.v.) [d-Pen2,5]enkephalin (DPDPE) and [d-Ala2]deltorphin II, agonists selective for δ1- and δ2-opioid receptors, respectively, was blocked following subcutaneous (s.c.) implantation of a naltrexone pellet (7.5 mg) for 7 days. Removal of the naltrexone pellet was followed 24 h later by a decrease of 7.5-fold in the ED50 value of [d-Ala2]deltorphin II, but not that of DPDPE. In a whole brain homogenate the binding of [][d-Ala2]deltorphin II was increased twice as much as that of []DPDPE. Chronic naltrexone treatment also produced an 8.6-fold decrease in the ED50 value of i.c.v. administered morphine. The increase in morphine potency was reversed to a control (placebo-treated mice) value by the selective δ2-opioid receptor antagonist, naltriben (25 pmol, i.c.v.). Thus, chronic naltrexone selectively increases δ2-opioid receptor-mediated antinociception, supporting the existence of δ opioid receptor subtypes with distinct adaptive characteristics. The data also indicate that δ2-opioid receptors are critically involved in the expression of morphine supersensitivity.
European Journal of Pharmacology.
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ABSTRACT: Studies in vivo demonstrate that antisense oligodeoxynucleotide (ODN) treatment specifically reduces the functions mediated by numerous central nervous system (CNS) receptors, including opioid receptors. However, the effects of antisense ODN on the opioid receptor mRNA target, itself are rarely examined. In the present study, the effect of supraspinal antisense ODN administration on δ-opioid receptor (DOR) mRNA levels in selected CNS regions, was investigated in mice. ODN targeting a 20-nucleotide sequence of the DOR mRNA transcript was administered by intracerebroventricular (i.c.v.) injection twice daily for 3 days. First, to confirm that antisense ODN treatment decreases DOR function in this system, antinociception produced by DOR-selective agonist [d-Ala2]deltorphin II was assessed on day 4. A 2-fold reduction in [d-Ala2]deltorphin II potency was revealed in antisense ODN-treated mice compared to mice receiving control treatments. DOR mRNA levels in selected CNS regions which either mediate antinociception; medial thalamus (MThal), periaqueductal gray (PAG), frontal cortex (FCtx) and spinal cord (SpC) or exhibit relatively high levels of DOR mRNA; nucleus accumbens (Acb) and caudate-putamen (CPu) were then quantitated by solution hybridization. Levels of DOR mRNA in antisense ODN-treated mice were not different from levels in mice treated with saline vehicle, which ranged from 0.07 pg/μg total RNA in MThal and PAG to 0.26 pg/μg total RNA in CPu. These results are both consistent with previous reports that antisense oligodeoxynucleotide (ODN) treatment down-regulates DOR protein in vivo and indicate that this down-regulation is not associated with altered DOR mRNA levels.
Molecular Brain Research.
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ABSTRACT: We have used a sensitive solution hybridization assay with a riboprobe transcribed from the coding sequence of the δ-opioid receptor gene (DOR) to study the up-regulation of the DOR mRNA by ethanol in NG108-15 cells. Exposure of the cells to compounds that increase cAMP levels (forskolin, forskolin+IBMX, or dibutyryl cAMP) resulted in the attenuation of ethanol-induced up-regulation of DOR mRNA. The inactive analogue of forskolin, 1,9-dideoxy forskolin had no effect. Northern blot analysis of RNA extracts from ethanol-, forskolin- or ethanol+forskolin-treated cells showed proportional changes in each of the multiple DOR mRNA bands, so that no difference was observed in the fraction of the total hybridization signal produced by each band of the DOR mRNA. In the absence of ethanol, forskolin or dibutyryl cAMP reduced the basal levels of DOR mRNA. The cAMP analogue (Rp)-cAMPS, a protein kinase A (PKA) inhibitor, increased DOR mRNA levels. However, the combination of (Rp)-cAMPS and ethanol did not further increase DOR mRNA levels compared to ethanol or (Rp)-cAMPS alone. Signaling through cAMP and PKA down-regulates DOR mRNA levels. The ethanol-induced increase in DOR mRNA levels in NG108-15 cells appears to be mediated via a reduction of PKA.
Molecular Brain Research.
